Characterization of the cellular and molecular bases of inborn errors of immunity

National Institute Of Allergy And Infectious Diseases

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Abstract

In FY2021, we have identified a new gene defect responsible for an X-linked combined immunodeficiency and contributed to the characterization of three other forms of inborn errors of immunity (IEI), two of which are due to newly identified gene defects. We have also expanded knowledge on the molecular and cellular bases and clinical features of known forms of IE. Furthermore, we have reported on the experience with newborn screening for severe combined imune deficiency (SCID) and illustrated some of the challenges associated with it. We have also participated at multi-center retrospective studies aimed at defining current outcome of hematopoietic stem cell transplantation (HSCT) in selected forms of IEI, demonstrating significant progress in the treatment of these disorders. We have provided pre-clinical evidence in vitro that gene editing may correct the cellular phenotype of one of the most severe forms of IEI, SCID due to RAG2 deficiency. Finally, we have reported on clinical outcome and development of adaptive immune responses after SARS-CoV-2 infection in some forms of IEI. In particular, the main results of the studies performed have been the following: 1) we have discovered a new X-linked combined immune deficiency due to mutations of the SASH3 gene, that encodes for an adaptor molecule. We have reported that this disease is characterized by susceptibility to severe and recurrent infections and treatment-refractory autoimmune cytopenias, associated with profound lymphopenia and neutropenia. Laboratory studies have shown that SASH3 plays a critical role in T-cell activation in response to TCR/CD3 engagement, and that SASH3 mutations are associated with increased apoptosis. These abnormalities have been reversed in vitro upon transfer of a normal copy of the SASH3 cDNA (publication #1). 2) By performing high-throughput sequencing of the TCR repertoire, we have contributed to define the cellular and molecular bases of two other novel forms of IEI, namely CD28 deficiency (publication #2) and PD-1 deficiency (publication #3). In particular, we demonstrated that TCR repertoire of these patients displayed normal diversity; this was also true for somatic revertants that were identified in patients with CD28 deficiency. These data have important implications on the mechanisms of intrathymic positive and negative selection of T cells, as they demonstrate that neither CD28 nor PD-1 play a critical role in these processes. We have 3) We also contributed to the characterization of the cellular and molecular phenotype of a patient with recalcitrant warts and rapidly progressing respiratory failure, due to mutation at the translation initiation site of the CD4 gene, leading to complete lack of CD4 surface expression (publication #4). Of note, we found that CD8+ T cells from this patient included MHC class II-restricted CMV-specific T cells, indicating tat compensatory mechanisms exist during thymic selection to endow CD8+ T cells with the capacity to recognize also MHC class II restricted antigens. 4) By performing in vitro T cell differentiation studies in a family with DNA polymerase delta 1 (POLD1) deficiency, we have demonstrated that this molecule plays acritical relent only in DNA repair but also in T cell development (publication #5) 5) By generating induced pluripotent stem cells from patients with TLR3 deficiency, followed by their targeted differentiation in vitro into cortical neurons, we have helped demonstrate that constitutive tonic TLR3 signaling inducing baseline type I IFN production controls antiviral immunity in central nervous system cells, and that defects of this pathway therefore lead to increased risk of herpes simplex encephalitis (publication #6). 6) We have conducted a series of studies that highlight the clinical, cellular and molecular heterogeneity of severe forms of IEI. These include the demonstration of bi-allelic ZAP70 mutations that lead to a SCID phenotype and yet retain the capacity of T cells to respond to mitogenic stimulation (publication #7), identification of an aberrant pattern of TCR alpha gene rearrangements in patients with TCR alpha constant chain (TRAC) gene deficiency (publication #8), and a series of publications illustrating the heterogeneity of clinical and laboratory features associated with RAG deficiency (publications #9-12).Besides generation of a reduced number of T cells (as observed in patients with hypomorphic RAG mutations), also partial impairment of signaling through the TCR/CD3 complex is a cause of autoimmunity, as demonstrated in patients with CD3 gamma (CD3G) deficiency (publication #13). 7) Autoimmunity is also a cardinal feature of Autoimmune Polyendocrinopathy Enteropathy Candidiasis Ectodermal Dystrophy (APECED), a condition characterized by high titers of autoantibodies targeting Inteferon-alpha (IFN_a) and IFN-omega (IFN-w). Consistent with the fact that these autoantibodies are associated with severe clinical curse in patients with COVID-19, we found that APECED patients are at high risk of poor outcome after SARS-CoV-2 infection (publication #14). Furthermore, we collaborated with the Lionakis Lab at NIAID to establish that increased IFN-gamma responses in patients and in a mouse model of APECED are a cause of chronic mucocutaneous candidiasis in this disease (publication #15). Nuclear factor kappa B2 (NFKB2) deficiency is another IEI with increased risk of autoimmunity. By studying the TCR repertoire of these patients, we have identified molecular signatures indicative of a self-reactive repertoire (publication #16). Finally, we explored other manifestations of immune dysregulation in IEI, including cerebellar ataxia and liver failure in patients with IPEX syndrome (publication #17) and nodular regenerative hyperplasia of the liver in patients with X-linked agammaglobulinemia (publication #18). T lymphocyte senescence is another mechanism contributing to imune dysregulation and impaired immunological surveillance against infections. By studying patients with CD40 ligand (CD40LG) deficiency, we have demonstrated that the CD8+ T cell compartment of these patients has signatures of cell senescence, which may contribute to the increased risk of infections in this disease (publication #19). 7) An important focus of our lab is the characterization of the cellular composition of the thymus in health and disease. We have used a mouse model of MHC-II deficiency to demonstrate that lack of MHC-II expression leads to perturbation of central and peripheral immunological tolerance (publication #20). In another study, we have defined that progressive T cell differentiation in the thymus is associated with finely regulated expression of phosphate transporters, uncovering a novel mechanism that regulates thymopoiesis (publication #21). 8) Since 2018, universal newborn screening for SCID is available across the country. We have reviewed the experience with this program, including its success and challenges (publications #22-24). 9) HSCT represents the mainstay of treatment for many IEI. We have reported on the success of HSCT for POMP deficiency (publication #25) and reviewed progress with HSCT for chronic granulomatous diseases and APDS (publications #26-27). 10) By performing gene editing in RAG2-mutated iPSCs, followed by targeted differentiation into T cells in vitro, we have produced proof of principle that gene editing can correct the cellular phenotype of the disease (publication #28). Similar results have bene obtained in MAGT1 deficiency (publication #29). 11) We have demonstrated that most patients with antibody deficiency retain the capacity to mount T- and B-cell responses to SARS-CoV2 (publication #30). 12) Finally, we have published several reviews on various aspects of IEI (publications #31-35).